The overall objective of this project is to define the cellular and molecular mechanisms of CD4 T-cell[unreadable] tolerance to a systemic self-antigen. The studies will test the hypotheses that cell-intrinsic tolerance in T-cells[unreadable] results from a combination of proximal signaling blocks and imbalanced cytokine production, and when the[unreadable] cell-intrinsic mechanisms of anergy and deletion fail, prolonged self-antigen recognition without other stimuli[unreadable] may lead to progressive development of regulatory T-cells. The studies will rely on a well-established[unreadable] transgenic model of systemic T-cell tolerance which has some unique strengths, notably that it is amenable[unreadable] to biochemical and molecular analyses of cells that have encountered self-antigen, and it is the only system[unreadable] in which effector and regulatory T-cells are generated sequentially from a monoclonal T-cell population in[unreadable] response to self-antigen recognition in peripheral tissues. The following specific aims will be addressed:[unreadable] 1. Mechanisms of cell-intrinsic tolerance (anergy and deletion) induced by a systemic self-antigen.[unreadable] These studies will define signaling blocks in T-cells rendered anergic by recognition of systemic self-antigen[unreadable] in vivo, using a novel multiplex phosphoprotein array and other techniques, and examine the roles of known[unreadable] T-cell regulators (CTLA-4, Fas, Bim) in systemic tolerance. In addition, the studies will explore the novel idea[unreadable] that imbalanced production of IFN-y without IL-2 contributes to T-cell tolerance, and define the mechanisms[unreadable] underlying this unexpected role of IFN-y as a tolerance-inducing cytokine.[unreadable] 2. Induction and functions of peripherally generated regulatory T-cells (Treg). Using a model of[unreadable] sequential development of effector T-cells and Treg in response to recognition of systemic antigen, these[unreadable] studies will define the lineage relationships between these two cell populations and the roles of cytokines in[unreadable] controlling the balance between effector and regulatory cells. A model of parent to F1 graft-vs-host reaction[unreadable] will be used to examine the development of effector and regulatory T-cells in situations of polyclonal T-cell[unreadable] reactivity.[unreadable] Thus, this project uses a defined experimental system and a variety of precise analytical methods to[unreadable] study fundamental mechanisms of peripheral T-cell tolerance, its induction and maintenance, and its[unreadable] regulation by external signals. There are numerous close interactions between this project and [unreadable] studies of CTLA-4 and Treg, signaling pathways, interactions of central and[unreadable] peripheral tolerance mechanisms. The results are not only of biological significance, but will also provide[unreadable] valuable leads for strategies to induce tolerance as a therapeutic modality.